Figure 1

Characterization of recombinant, eGFP-expressing guinea pig cytomegalovirus (GPCMV) used for in vivo antiviral studies. An eGFP-expressing, recombinant GPCMV was generated using gpt selection to enable detection of infection in the guinea pig model of congenital CMV infection [11]. Panel A, genome map of GPCMV genome (Hin d III digestion profile) indicating site of insertion of eGFP/gpt cassette. Insertion of this cassette into GPCMV genome did not disrupt any predicted conserved CMV open reading frames, based on homology comparisons with other CMVs and BLAST search [6]. Panel B, fluorescence microscopy analysis of vAM403 virus in tissue culture. GPL cells were inoculated with recombinant virus and visualized by FITC filter 96 hours post-infection. Panel C, analysis of vAM403 protein expression and comparison to wild-type GPCMV. GPL cells infected with either wild-type virus (lanes 1, 3) or vAM403 (lanes 2, 4) were labelled with ³⁵S-methionine/cysteine, and immunoprecipitated with either polyclonal anti-GPCMV antisera (lanes 1, 2) or a monoclonal antibody against eGFP (lanes 3, 4). Position of molecular weight markers is indicated. Precipitation with serum from a GPCMV seronegative guinea pig did not precipitate any viral proteins (data not shown). The pattern of proteins recognized by immune serum against wild-type and vAM403 virus is virtually identical, with exception of a band of ~85 kDa present in wild-type infected cells, but not present in vAM403-infected cells (arrow). Known positions of migration of gB complex (gp90 and gp58) are indicated, as is the position of migration of UL83 homolog (arrowheads). The eGFP monoclonal antibody precipitated the ~30 kDa eGFP protein from vAM403 cells (lane 4, arrowhead), but not wild-type cells (lane 3).